Team:METU Turkey/Results Discussion/CooA Expression and Purification

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<p style="font-size:110%; color:#576f91; font-family:georgia,serif;"><br>  
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<br> <h2>1-Overexpression optimization</h2>
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<br>Fermentor studies
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<br> <h3>A. Expression vector comparison (pKK versus pTriEx)</h3>
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<br>When we compare the below results, the expression of pTriEx vector is higher than the expression of pTriEx vector
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<br>- Culturing: These studies showed that our E.coli BL21 DE3 strain grow well until absorbance at 600 nm reaches 2.2. However, because of high growing capacity of fermentor, sufficient time is not present for Carbon monoxide induction. Therefore, we changed fermentor to flask in order to have higher induction time period of carbon monoxide
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<br> <h3>A.1. Expression in BL21 with pTriEx</h3>
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<br>- Viability: Cells having absorbance at 0.05 have reached the stationary phase in 3 hours and starting from 4th hour, our bacteria cultures started to die.
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<br>- Reproducibility: Our data acquired from fermentor studies are not reproducible because CO was induced to death cells.
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<div align="center" style="margin:15px 0px 0px 0px"><img style="border: 0px solid ; width: 300px; height: 300px;" alt="w7" src="https://static.igem.org/mediawiki/2010/3/3b/Growth_curve_alpha_1.JPG"></a></div>
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<br> In this expression experiment, we obtained 2 different bands in BL21 pTriEx from BL21 negative control as indicated gel photo. CooA is found in two form (monomer and dimer).
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<br>- Myoglobin Assay: Our data acquired from myoglobin assay is not clear because absorbance acquired from cell density have interefered with myoglogin assay data.
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<div align="center" style="margin:15px 0px 0px 0px"><img style="border: 0px solid ; width: 400px; height: 400px;" alt="w7" src="https://static.igem.org/mediawiki/2010/3/35/Br1.jpg"></a></div>
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<br>- CFU(Colony Forming Unit): We couldn’t observe growth of bacteria starting from 4th  hour of fermentation.
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<br>- Fluorescence measurements: Fluorescence was detected due to auto-fluorescence of CooA proteins. Since our bacteria cultures have expression vector, CooA is highly expressed which in turn causes fluorescence.</br>
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<br> <h3>A.2. Expression in JM109 and BL21 with pKK vector</h3>
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We failed to express pKK vector in JM109. We have a little expression in BL21.
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<br>Flask experiments</br>
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<div align="center" style="margin:15px 0px 0px 0px"><img style="border: 0px solid ; width: 400px; height: 400px;" alt="w7" src="https://static.igem.org/mediawiki/2010/b/b9/Br2.jpg"></a></div>
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<br>  <html><div><a href="https://2010.igem.org/Team:METU_Turkey/Results_Discussion/pKK_versus_pTriEx"><strong>Details...</strong></a></div></html>
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<br>- Culturing: We didn’t have enough time to test our final vector. Instead, we have used BBa_K352017 to test the RFP production. We have used 300 mL flasks with 80 mL LB culture. We have applied 80 Ml Carbon monoxide to some. The results were successful.  Cultures induced with carbon monoxide successfully expressed red fluorescent protein.
 
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<div align="center" style="margin:15px 0px 0px 0px"><img style="border: 0px solid ; width: 300px; height: 300px;" alt="w7" src="https://static.igem.org/mediawiki/2010/4/49/Culturing_alpha_1.JPG"></a></div>
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<br> <h3>B. Expression host comparison (JM109 versus BL21)</h3>
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<br> BL21 is better host than JM109 for CooA.
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<br> <h3>C. IPTG induction studies</h3>
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<br>Myoglobin assay optimization</br>
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<br> Expression comparison with different IPTG Concentration of pTriEx vector.
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<html>
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<div align="center" style="margin:15px 0px 0px 0px"><img style="border: 0px solid ; width: 400px; height: 400px;" alt="w7" src="https://static.igem.org/mediawiki/2010/6/6a/Br3.jpg"></a></div></html>
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<br> Our CooA pTriEx vector is expressed without IPTG induction but is less than others. When 2mM IPTG was added to culture, expression of CooA was decreased. It has probably a toxic effect on our bacteria.
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<br>- We have planned to measure CO presence with myoglobin assay. We used myoglobin purchased from Sigma to detect levels of Carbon monoxide. In our experiments, we couldn’t measure exact CO in the environment owing to the fact that sensitivity level of the myoglobin bonded with CO was not sufficient to observe absorbance at 540 nm.
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<br> <html><div><a href="https://2010.igem.org/Team:METU_Turkey/Results_Discussion/IPTG_induction"><strong>Details...</strong></a></div></html>
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Signal quantification with fluorescence spectroscopy
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<br> <h3>D. Ferric Citrate effect</h3>
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<br>Signal Quantification of pCooF-RBS-GFP-TT: </br>
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<br> Time Dependent Expression of pTriEx vector  with Ferric Citrate and without Ferric Citrate
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<html>
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<div align="center" style="margin:15px 0px 0px 0px"><img style="border: 0px solid ; width: 400px; height: 400px;" alt="w7" src="https://static.igem.org/mediawiki/2010/5/5f/Br4.jpg"></a></div></html>
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<br>For this experiment, the bacteria containing the vector having the construct and expression vector pTriEx is incubated in E.coli BL21 strain at different conditions. The results are as the following;
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<br> According to the SDS-Page result, Ferric Citrate increses the expression of CooA. It is probably releated to heme characteristic of CooA. The best IPTG induction time among 3-5-7-9 hours is 5 hour.
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<div align="center" style="margin:15px 0px 0px 0px"><img style="border: 0px solid ; width: 300px; height: 300px;" alt="w7" src="https://static.igem.org/mediawiki/2010/1/1b/PCooF_experiment_alpha_1_florasan_grafik.JPG"></a></div>
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<br>  <html><div><a href="https://2010.igem.org/Team:METU_Turkey/Results_Discussion/Ferric_Citrate_effect"><strong>Details...</strong></a></div></html>
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<br> <h3>E. Soluble versus insoluble fraction</h3>
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<br>- From the data acquired from spectrophotometer, we observed that neither CO presence nor the presence of pCooF-RBS-GFP-TT affected GFP intensity. We acknowledged that fluorescence increase in the intensity of 510 nm was result of fluorescent nature of CooA. It is suspected that the result is dependent on our strain, BL21, caused the unexpected results. 
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Signal Quantification of pCooM-RBS-RFP-TT-pLac-RBS-CooA: We have tried another construct pCooM-RBS-RFP-TT-pLac-RBS-CooA at TOP10 strain. The results were successful. We have put our construct in different environments (flasks with different conditions) and compared the RFP fluorescence.
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<div align="center" style="margin:15px 0px 0px 0px"><img style="border: 0px solid ; width: 300px; height: 300px;" alt="w7" src="https://static.igem.org/mediawiki/2010/1/1b/PCooF_experiment_alpha_1_florasan_grafik.JPG"></a></div>
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<br> Lysis Buffer with Urea and without Urea
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<div align="center" style="margin:15px 0px 0px 0px"><img style="border: 0px solid ; width: 400px; height: 400px;" alt="w7" src="https://static.igem.org/mediawiki/2010/4/43/Br5.jpg"></a></div></html>
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</br><br>
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<br> Results are seen in the SDS-Page photograph, there is no difference between bands of same samples (look at all the 50kDa bands). Thus,we concluded that CooA does not accumulate in inclusion body.
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Imaging with confocal laser scanning microscopy</br>
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<br>  <html><div><a href="https://2010.igem.org/Team:METU_Turkey/Results_Discussion/Soluble_vs_insoluble_fraction"><strong>Details...</strong></a></div></html>
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<br> Signal Visualization of pCooM-RBS-RFP-TT-pLac-RBS-CooA:
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<br> <h2>2-Purification optimization</h2>
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<div align="center" style="margin:15px 0px 0px 0px"><img style="border: 0px solid ; width: 300px; height: 300px;" alt="w7" src="https://static.igem.org/mediawiki/2010/2/26/Confocal_sa%C4%9F.JPG "></a></div>
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<br> <h3>A. Ion exchange chromatography with Sepharose Q (QS)</h3>
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<div align="center" style="margin:15px 0px 0px 0px"><img style="border: 0px solid ; width: 300px; height: 300px;" alt="w7" src="https://static.igem.org/mediawiki/2010/a/ae/Confocal_sol.JPG "></a></div>
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<br> Purification QS with BL21 pTriEx: Yellowish color indicates our CooA protein. Then we looaded 27,28,29,30 and 31 to SDS-Gel the elutes which have a high value at 420nm because oxidized CooA gives a Sorret peak at 420 nm according to Aono. CooA concentration was determined according to extinction coefficient of heme (Ferric State) which is 108 mM-1 cm-1 according to Aono. The SDS gel indicates that our protein is colose to 50 kDa in dimer. MW of monomer is estimated 24-25kDa according to Aono.
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<br>  <html><div><a href="https://2010.igem.org/Team:METU_Turkey/Results_Discussion/QS_results"><strong>Details...</strong></a></div></html>
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<div align="center" style="margin:15px 0px 0px 0px"><img style="border: 0px solid ; width: 400px; height: 400px;" alt="w7" src="https://static.igem.org/mediawiki/2010/1/14/Br13.jpg"></a></div></html>
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<br> <h3>B. Affinity chromatography with chealating sepharose(CS)</h3>
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<br> Purification with CS BL21 pTriEx: Loaded combined FPLC tubes from QS column(10/09/26) gave two close picks in CS column. First one corresponds to the CooA. The elutions of CooA are C13,C14 and C15. We combined them for further analyzes with EMSA and ITC and stored @+4. C16 AND C17 are combined to reload to CS column because they are contaminated another protein which is close to 40kDa.
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<br>  <html><div><a href="https://2010.igem.org/Team:METU_Turkey/Results_Discussion/CS"><strong>Details...</strong></a></div></html>
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<html>
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<div align="center" style="margin:15px 0px 0px 0px"><img style="border: 0px solid ; width: 400px; height: 400px;" alt="w7" src="https://static.igem.org/mediawiki/2010/7/74/Br15.jpg"></a></div></html>
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<div align="center" style="margin:15px 0px 0px 0px"><img style="border: 0px solid ; width: 400px; height: 400px;" alt="w7" src="https://static.igem.org/mediawiki/2010/d/d8/Br16.jpg"></a></div></html>
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<br> <h2>C. Concentrated CooA</h2>
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<br> CooA protein contains heme group therefore its solution is seen yellow to orange
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<div align="center" style="margin:15px 0px 0px 0px"><img style="border: 0px solid ; width: 400px; height: 400px;" alt="w7" src="https://static.igem.org/mediawiki/2010/b/be/Br18.jpg"></a></div></html>
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Latest revision as of 03:39, 28 October 2010

Home

 


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3-CooA Expression and Purification



1-Overexpression optimization


A. Expression vector comparison (pKK versus pTriEx)


When we compare the below results, the expression of pTriEx vector is higher than the expression of pTriEx vector

A.1. Expression in BL21 with pTriEx


In this expression experiment, we obtained 2 different bands in BL21 pTriEx from BL21 negative control as indicated gel photo. CooA is found in two form (monomer and dimer).
w7

A.2. Expression in JM109 and BL21 with pKK vector

We failed to express pKK vector in JM109. We have a little expression in BL21.
w7



Contents

B. Expression host comparison (JM109 versus BL21)


BL21 is better host than JM109 for CooA.


C. IPTG induction studies


Expression comparison with different IPTG Concentration of pTriEx vector.

w7


Our CooA pTriEx vector is expressed without IPTG induction but is less than others. When 2mM IPTG was added to culture, expression of CooA was decreased. It has probably a toxic effect on our bacteria.



D. Ferric Citrate effect


Time Dependent Expression of pTriEx vector with Ferric Citrate and without Ferric Citrate

w7


According to the SDS-Page result, Ferric Citrate increses the expression of CooA. It is probably releated to heme characteristic of CooA. The best IPTG induction time among 3-5-7-9 hours is 5 hour.



E. Soluble versus insoluble fraction


Lysis Buffer with Urea and without Urea

w7


Results are seen in the SDS-Page photograph, there is no difference between bands of same samples (look at all the 50kDa bands). Thus,we concluded that CooA does not accumulate in inclusion body.



2-Purification optimization


A. Ion exchange chromatography with Sepharose Q (QS)


Purification QS with BL21 pTriEx: Yellowish color indicates our CooA protein. Then we looaded 27,28,29,30 and 31 to SDS-Gel the elutes which have a high value at 420nm because oxidized CooA gives a Sorret peak at 420 nm according to Aono. CooA concentration was determined according to extinction coefficient of heme (Ferric State) which is 108 mM-1 cm-1 according to Aono. The SDS gel indicates that our protein is colose to 50 kDa in dimer. MW of monomer is estimated 24-25kDa according to Aono.



w7

w7

w7


B. Affinity chromatography with chealating sepharose(CS)


Purification with CS BL21 pTriEx: Loaded combined FPLC tubes from QS column(10/09/26) gave two close picks in CS column. First one corresponds to the CooA. The elutions of CooA are C13,C14 and C15. We combined them for further analyzes with EMSA and ITC and stored @+4. C16 AND C17 are combined to reload to CS column because they are contaminated another protein which is close to 40kDa.



w7

w7



C. Concentrated CooA


CooA protein contains heme group therefore its solution is seen yellow to orange

w7




-- </br>

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